Last year we showed by antibody-staining of fixed cells that endogenous DMIB is localized uniformly on the plasma membrane of resting cells, at active protrusions and cell-cell contacts of randomly moving cells, and at the front of motile polarized cells. The phospohlipid-binding BH site in the tail is both required and sufficient for association of DMIB with the plasma membrane at all stages and all the membrane regions enriched in DMIB were also enriched in either or both PIP2 and PIP3. However, although required for plasma membrane association, the BH-site alone is not sufficient for proper relocalization of DMIB during random cell movement and starvation-induced cell polarization since localization of tail alone was different than localization of full length myosin. In all situations that we have studied, there were two main factors determining DMIB localization: binding to plasma membrane lipids through the BH-site in the tail and binding to cytoplasmic F-actin through the ATP-sensitive actin-binding site in the head. These two major interactions competing with each can explain most of the differences between the localization of expressed tail and full-length DMIB. Last year, we observed, and published (Brzeska et al., 2012), a strong association of the strong actin-binding mutant (DMIB-N154A) with structures resembling actin waves. Actin waves are F-actin structures self-propagating through the cell by a treadmilling mechanism, i.e. addition of actin monomers to the plus end and dissociation of monomers from the minus end of actin filaments. DMIB is a known component of actin waves but how DMIB associates with actin waves and DMIBs role in the formation and function of actin waves are not known. We have now developed a system for co-transfection of Dictyostelium with GFP-tagged DMIB mutants and RFP-labeled F-actin marker (actlife). This allows us to monitor the dynamic localization of F-actin and DMIB in live wild-type and DMIB-null cells. We confirmed by TIRF microscopy that the strong actin-binding mutant DMIB-N154A associates with actin waves. We found that actin-binding by the DMIB head is absolutely required for association of DMIB with actin waves since the weak actin-binding DMIB mutant (DMIB-E407K) does not associate with waves. We also found that, under certain conditions, DMIB-N154A (the strong actin-binding mutant) enhances formation of actin waves. However, DMIB is not absolutely required for wave formation since DMIB-null cells form waves. In one current model of the function of actin waves, DMIB binds to the plasma membrane via the lipid-binding site in its head and anchors the actin wave to the plasma membrane (and also possibly recruits proteins involved in actin polymerization) via its tail. We are testing this hypothesis by expressing in DMIB-null cells DMIB mutants with a compromised lipid-binding site in the head and/or a compromised SH3 domain (that interacts with other proteins involved in actin polymerization) in the tail. The experiments are in progress with no clear results at this time. It has also been proposed that DMIB acts within the actin wave as a plus-end motor displacing actin filaments from the plasma membrane thus allowing addition of actin monomers to the elongating actin filament. Our results with the strong actin binding mutant, which does not have motor activity, are not consistent with this proposal.